microRNAs (miRNAs) are small RNAs known to be necessary for normal development
of vertebrates, including development of the heart. miRNAs act primarily through
post-transcriptional inhibition of protein expression. Two miRNAs described in this thesis, miR-138 and miR-202*, are shown to be necessary for cardiac development in zebrafish.
miR-138 ensures proper patterning of the cardiac chambers by clearing mRNA
transcripts from the ventricle, allowing proper maturation of ventricular
cardiomyocytes. Reduction in miR-138 results in cardiac dysfunction and lack of maturation in cardiomyocytes located on the outer curvature of the ventricle. Direct inhibition of Raldh2 protein by miR-138 in the ventricle, resulting in reduced expression of retinoic acid (RA), is necessary to restrict expression of proteins such as Versican to the atrioventricular canal. Interestingly, Versican expression in the ventricle is also directly inhibited by miR-138. Thus miR-138 is demonstrated to be the first miRNA involved in cardiac patterning, and achieves inhibition of Versican expression both directly and indirectly via targeting of Raldh2. The targeting of multiple members of a pathway allows for robust clearance of early ventricular mRNAs.
miR-202*, in contrast, is necessary for both proper development and function of
zebrafish hearts. Inhibition of miR-202* in zebrafish embryos leads to
randomization of the left-right patterning of the heart, as well as cardiac conduction abnormalities. In cell culture, miR-202* is able to target Pkd2 protein for inhibition; Pkd2 is a calcium channel known to be necessary for specification of the left-right
axis in vertebrates. Embryos with knockdown of miR-202* also demonstrate a 2:1 atrioventricular node (AV) block, where the atrium conducts twice for every ventricular conduction. The molecular mechanism for 2:1 AV block remains unknown, but elucidating the targets responsible for the phenotype will give insight into human diseases with similar conduction abnormalities.
In summary, the studies described here identify two miRNAs as being necessary
for normal development and/or function of the heart. Future studies further
elucidating the role of miR-138 and miR-202* in mammals will provide insight into connections between these miRNAs and human disease.